Host status of melon, carrot, and Meloidogyne incognita-susceptible and -resistant cotton, cowpea, pepper, and tomato for M. floridensis from California

Abstract The host status of carrot, melon, and susceptible and resistant cultivars of tomato, cotton, cowpea, and pepper for a California isolate of the peach root-knot nematode Meloidogyne floridensis was determined in greenhouse pot experiments. It was compared to a race 3 isolate of M. incognita. Melon was an excellent host for both isolates and roots were heavily galled after the 8-week trial. Carrot was a host for M. incognita, but a poor host for M. floridensis, although both isolates caused similar levels of galling. Susceptible cotton was a good host for M. incognita race 3, but a poor host for M. floridensis. Susceptible tomato, cowpea, and pepper were good hosts for both isolates. The M. incognita resistance in tomato and pepper was broken by M. floridensis. Resistant cowpea was a maintenance host as population levels of M. floridensis remained virtually unchanged over the trial period. We conclude that M. floridensis poses a risk to some important vegetable crops in California, as it reproduces on most vegetable crops, including some cultivars that are resistant to M. incognita. On susceptible crops, the reproduction of M. floridensis was always significantly less than that of M. incognita, and we hypothesize that in mixed species field populations, M. incognita will outcompete M. floridensis. This study demonstrates that efforts to limit the spread and prevent further introductions of M. floridensis in California are important to maintain the effectiveness of plant resistance as a nematode management strategy in vegetable crops.

Root-knot nematodes (RKN, Meloidogyne spp.) are economically important plant parasites with a broad host range (Luc et al., 1990;Castagnone-Sereno et al., 2013).The Southern root-knot nematode (M.incognita) is a common RKN in California, particularly in loamy and sandy soils.Host crops for this nematode include bell pepper (Capsicum annuum), carrot (Daucus carota), cantaloupe/ honeydew melon (Cucumis melo), and tomato (Solanum lycopersicum) (Luc et al., 1990).All these are economically important crops in California.The state is the leading producer of each of these crops in the U.S.A., representing a combined acreage of 352,000 acres and a combined value of $2,232 million (CDFA, 2022).Cotton (Gossypium barbadense/Gossypium hirsutum) is grown on 113,00 acres in California with a value of $400 million (CDFA, 2022) and is a host to M. incognita race 3 and 4 populations (Taylor and Sasser, 1978).To limit crop damage and prevent RKN population build-up, host plant resistance can be an excellent management strategy.It reduces or eliminates the need for nematicides and does not require major changes in farming practices or purchasing special equipment.Host-plant resistance has been adopted on a large scale in California processing tomato production, and the vast majority of current cultivars are resistant to M. incognita, M. javanica, and M. arenaria (Roberts, 1992;Williamson, 1998).Although the resistance in all tomato cultivars is based on the presence of the single dominant Mi-1 gene (Kaloshian et al., 1996;Williamson, 1998) that has been employed for over 70 years, and resistance-breaking RKN populations occur in California and elsewhere (Williamson, 1998;Ploeg et al., 2023), it remains an effective management strategy (Castagnone-Sereno et al., 2007).Resistant bell pepper cultivars containing the N-gene conferring resistance to M. incognita (Thies et al., 2003) are available but have yet to be evaluated under commercial field conditions in California.Meloidogyne incognita-resistant cotton cultivars are available and are commercially grown (Ogallo et al., 1997;Ojeda-Rivera et al., 2022).A breeding program to develop M. incognita-resistant carrot cultivars is ongoing (Seo et al., 2014;Parsons et al., 2015;Roberts et al., 2016), but resistant carrot cultivars are not (yet) available.Similarly, all melon cultivars are susceptible to M. incognita (Nugent and Dukes, 1997).Although RKN resistant Cucumis species, particularly C. metuliferus, can be used as a rootstock for melon, they are not commercially available, which may negatively affect fruit quality (Siguenza et al., 2005;Exposito et al., 2020), and this strategy is not used in California melon production.Cowpea (Vigna unguiculata) is also highly susceptible to M. incognita (Roberts et al., 2005), and grain-type cultivars are grown as dry blackeye beans or peas in California on 3,500 acres (CDFA, 2022).However, different, vigorous growing, late blooming vining-type cultivars of cowpea are also of interest as a cover crop that can shade out weeds, fix nitrogen, and add a substantial amount of biomass to the soil (Aguiar et al., 2001;Roberts et al., 2005).A third type of cowpea (Vigna unguiculata subsp.sesquipedalis), known as long bean or asparagus bean, is grown in central California, mostly on small-scale diversified farms by Hmong growers for fresh consumption of the beans (Huynh et al., 2023).Cultivars of all three types of cowpea with Rk-gene resistance against M. incognita have been developed (Roberts et al., 2005;Huynh et al., 2023).They can be used to minimize nematode-associated yield loss (blackeye bean, long bean) and, when used as a cover crop, can also reduce soil nematode levels for a subsequent crop (Roberts et al., 2005;Huynh et al., 2023).
The peach root-knot nematode (M.floridensis) was found for the first time in California in 2018 from soil and root samples collected from stunted almond trees grafted onto a rootstock with resistance to the Southern root-knot nematode M. incognita in Merced County CA, USA (Westphal et al., 2019).Root samples collected from these resistant rootstocks exhibited galling symptoms typical for RKN infestation, and morphological and molecular analysis of RKN extracted from root and soil samples confirmed the presence of the peach root-knot nematode M. floridensis (Westphal et al., 2019).As a consequence of this first detection of M. floridensis from California, the nematode is now considered an A-rated pest, which means that any additional finds would also be subject to treatment and eradication efforts.Cultures of this nematode can only be kept in a USDA-certified quarantine facility (Chitambar, 2018).This nematode appears to have a very limited distribution in California, as subsequent preliminary sampling efforts have not resulted in positive findings.In other US states, the nematode has been reported from Florida (Brito et al., 2015), South Carolina (Reighard et al., 2019), and Georgia (Marquez and Hajihassani, 2023).The host range and morphology of M. floridensis appear similar to M. incognita.In the differential host test (Taylor and Sasser, 1978), the plants may respond similarly to M. incognita race-3 and M. floridensis (Marquez and Hajihassani, 2023).Consequently, M. floridensis was initially considered a race-3 isolate of M. incognita (Sherman and Lyrene, 1983;Handoo et al., 2004).However, a species-specific primer set (C2F3/M-Flor-R2) developed to amplify a short mitochondrial DNA fragment of M. floridensis has proven very useful in distinguishing this species from other common RKN species, i.e., M. incognita, M. javanica, and M. arenaria (Marquez and Hajihassani, 2023).Isolates of M. floridensis from Florida, South Carolina, California, and Georgia were genetically very similar (Marquez and Hajihassani, 2023).However, this was not reflected in the reproduction of different isolates on host plants.Limited data are available on the host status of vegetable crops for M. floridensis, and results differed among various studies or M. floridensis isolates.In multiple studies, tomato, eggplant, and watermelon were reported as hosts, and collards and peanuts as poor or nonhosts (Table 1).Cucumber and pepper were reported as a host or as poor/non-hosts depending on the study (Table 1).The reproduction factor RF (Pf/Pi = final population density/initial population density) was greater than 1 for M. floridensis on tomato cultivars containing the Mi-1 resistance gene in two of three studies (Table 1).The N-gene M. incognita-resistant pepper cultivar Charleston Belle was a poor host or a host for M. floridensis in the same study, depending on the M. floridensis isolate (Table 1).
The California isolate of M. floridensis was reported to break resistance in Mi-1 gene carrying tomato and in some sweet potato cultivars resistant to M. incognita (Ploeg and Stoddard, 2023).Still, it is unknown if this isolate can also break resistance in other vegetable crops.In this study, we compared the reproduction and root-galling of the California isolate of M. floridensis to an M. incognita race-3 isolate on M. incognita-susceptible and -resistant tomato, pepper, and cowpea.Although no resistant cultivars are available, melon and carrot were included because their host status for M. floridensis is unknown, and they are important RKN-susceptible vegetable crops in California.Cotton was included as an important non-vegetable crop with RKN-resistant cultivars available.

Materials and methods
Meloidogyne isolates: The isolation, culturing, and identification of the California Meloidogyne floridensis isolate (Mflor) and the California  (seed kindly supplied by Dr. Huynh, Dept.Nematology, UC Riverside) was determined in a replicated quarantine-greenhouse pot study.Tomato and pepper were seeded in a multicell seedling tray with potting mix (Sunshine Mix 5, Sungro, Vancouver, BC, Canada) and placed in a greenhouse.Three (tomato) or four (pepper) weeks after emergence, plants were carefully removed from the seedling trays, and roots gently washed free of potting mix and transplanted into 1-liter white plastic cups filled with steam-sterilized sand (93% sand, 4% silt, 3% clay, pH 7.1).Cotton, carrot, cowpea, and melon were seeded directly in 1-liter white plastic cups filled with steam-sterilized sand (three seeds per cup) and thinned to leave one plant per cup one week after emergence.Ten pots were set up for each crop cultivar, and five pots were each inoculated with 10 ml of a suspension containing 1,000 eggs/ml of Mflor and five pots with Minc-3 by adding 2.5 ml to each of four holes around the roots of each plant (10,000 eggs per plant).All pots were inoculated at the same time (tomato and pepper 1 week after transplanting, carrot 4 weeks after emergence, cotton 3 weeks after emergence, cowpea and melon 2 weeks after emergence).The pots were randomized over a metal galvanized greenhouse bench, allowing free water draining.They were watered daily with a slow drip to avoid water/soil splashing contamination among pots through an automated drip-irrigation system and fertilized one week after inoculation with 5 g of Osmocote 17-6-10 controlled release fertilizer (Scotts-Sierra Horticultural Products Co, Marysville, OH).The greenhouse temperature ranged between 21-26 o C under natural light.Plants were removed after 8 weeks, and the roots were washed free of soil.They were rated for the presence of root galling (0-10 scale: 0=no galling, 10=100% of root system galled.Bridge and Page, 1980).Eggs were extracted from the roots by shaking for 3 min in a 0.5% NaOCl solution (Hussey and Barker, 1973), collected by washing over two stacked 25 µm pore-size sieves, and counted under a dissecting microscope at 40x magnification.The complete experiment was repeated once.
The effect of the two RKN isolates on rootgalling and egg production was analyzed separately for each plant cultivar.Prior to analysis, egg counts were transformed by x 1 = log10(x+1) to standardize the variance.The significance of the RKN isolate, the repeated experiment (1 or 2), and the interactive effect on egg production was determined using the ANOVA procedure in SAS statistical software.Where treatment effects were significant at the 95% confidence level, treatment means were compared using Fisher's LSD procedure also at the 95% confidence level.Effects on the average galling index were analyzed with the non-parametric Kruskall-Wallis test (SAS Institute, Cary, N.C.).

Results
The interactive effect of "experiment x RKN isolate" on the degree of root galling was significant (P < 0.05) for melon, the susceptible tomato, the resistant pepper, and the susceptible and resistant cowpea.

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Therefore, the results are shown separately for the two experiments (Table 2).
Roots of melon Durango and the susceptible tomato Daniela were heavily galled, and roots of the resistant cowpea CB46RR remained virtually free of galling after inoculation with either Meloidogyne isolate.Mflor resulted in consistently higher root galling than Minc-3 on the resistant tomato Celebrity and the resistant pepper Carolina Wonder.However, the reverse was true with Minc-3 on the susceptible pepper Baron, resulting in more severe galling than Mflor.
Statistical analysis of the nematode reproduction x 1 = [log 10 (eggs per root system + 1)] showed that the "experiment x RKN isolate" interactive effect was only significant on the susceptible cowpea CB46Null (results not shown).However, as the separation of the means was the same between the two experiments, the results are presented as the average of 10 replicates.
The host status of a crop cultivar for RKN can be categorized according to the RF of the nematode on that plant as a good host (RF ≥ 1), a poor host (0.1 < RF <1), or a non-host (RF ≤ 0.1) (Oostenbrink, 1966;Sasser et al., 1984).The reproduction factor RF was significantly different between Mflor and Minc-3 on each of the tested crop cultivars (Table 3).On the susceptible crop cultivars tomato Daniela, cowpea CB46Null, pepper Baron, and melon Durango, RF values for Minc-3 were significantly higher than for Mflor, but as RF values on all these crop cultivars were much greater than 1 (RF values: 6.3 -57.2), they can be considered as good hosts for both RKN isolates (Table 3).The resistance of cowpea CB46RR, pepper Carolina Wonder, and tomato Celebrity to Minc-3 was very strong, with RF values of 0.1, 0.1, and 0.15, respectively.However, Mflor was able to overcome the resistance in tomato and pepper, instead increasing 1.8-fold and 8.2-fold, respectively, on these resistant crop cultivars (Table 3).The resistance in cowpea to Mflor was slightly compromised, and population levels on this crop stayed approximately even over the duration of the trial (RF = 0.9).Both cotton SJ2 and carrot Imperator 58 were good hosts for Minc-3 but poor hosts for Mflor (Table 3).Surprisingly, this difference was not reflected in root-galling (Table 2).

Discussion
This is the first report on the host status of carrot and melon for M. floridensis.Carrot has been reported in many studies as a good host for four common RKN species: M. incognita, M. javanica, M. arenaria, and M. hapla (Ferris, 2023).Race 1 isolates of the economically important Columbia RKN M. chitwoodi reproduce on carrots and can be distinguished from race 2 isolates, which do not reproduce on this crop (Mojtahedi et al., 1988;Santo et al., 1988).In this study, carrot was a poorer host for M. floridensis (RF = 0.7) than for M. incognita (RF = 3.6).
According to these RF values, carrot is designated as a poor host for M. floridensis and as a good host for M. incognita.Whether large differences exist in the host status of carrots for different isolates of M. floridensis remains to be studied.Interestingly, the ability to induce galling on carrots was not different between M. floridensis and M. incognita.Therefore, it is likely that growing carrots on fields infested with M. floridensis will impact carrot quality similarly to M. incognita.Like carrot, melon is also a good host for the most common RKN species (Ferris, 2023).In California, M. incognita is the most important RKN species on melon, resulting in stunting, yield loss, and severe root galling (Radewald et al., 1975).In this study, melon was an excellent host for both M. incognita and M. floridensis, although reproduction of the former was higher (RF 18.0 vs. RF = 9.5).Melon roots were severely galled after inoculation with either RKN isolate.Results on tomato corresponded with our previous results (Ploeg and Stoddard, 2023).The susceptible Daniela is an excellent host for both RKN isolates and the Mi-gene resistant Celebrity is resistant

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to M. incognita but susceptible to M. floridensis.The ability of M. floridensis to break Mi-gene resistance in tomato also agrees with previous results (Stanley et al., 2009;Marquez and Hajihassani, 2023).Tomato root-galling caused by M. floridensis was light but still significantly higher than after M. incognita inoculation.It can, therefore, be concluded that Mi-gene-carrying tomato cultivars allow the reproduction of M. floridensis and that attempts to use host plant resistance as a management strategy to lower M. floridensis population levels will probably prove ineffective in tomatoes.Cotton SJ2 was a good host for the M. incognita isolate used in this study.This was expected as the isolate was a race 3, able to reproduce on cotton (Taylor and Sasser, 1978).Handoo et al. (2004) reported that cotton was slightly infected and rated it a poor host for M. floridensis.This agrees with our results, as cotton was infested, and the roots developed slight galling, but the population declined (RF = 0.7).The resistant cotton DP 1747NR B2XF was resistant to M. incognita and highly resistant to M. floridensis, and using RKNresistant cotton promises to be an effective method for lowering both M. incognita and M. floridensis populations.Nyczepir, 2004), while others reported pepper as a host (Stanley et al., 2009).Variability between isolates of M. floridensis or differences in experimental conditions or between pepper cultivars used may be the reason for the reported differences in the host status of pepper for M. floridensis.The N-gene resistance in pepper Carolina Wonder was very effective against M. incognita but ineffective against M. floridensis.Stanley et al. (2009) reported RF values ranging between 0.3 and 1.2 of four M. floridensis isolates from Florida on the N-gene resistant pepper Charleston Belle.They concluded that this pepper cultivar was susceptible to two and resistant to two isolates but that even the two poorly reproducing isolates were able to overcome the N-gene resistance.Compared to their results, the results from our experiments are much less ambiguous as the N-gene-carrying Carolina Wonder can be designated as a good host for M. floridensis.Interestingly, the reproduction of M. incognita was significantly higher than that of M. floridensis on all the susceptible crop cultivars.This would imply that, under temperatures similar to those in our experiments, a field population consisting of a mixture of M. incognita and M. floridensis under a susceptible  FlordaGuard, Guardian, Hansen 536, and Bright's Hybrid 5 (Handoo et al., 2004;Stanley et al., 2009;Westphal et al., 2019).
In conclusion, our results show that M. floridensis is able to overcome resistance in pepper, tomato, and, to a lesser extent, cowpea and that some important California vegetable crops are a host for this nematode (Table 4).
The nematode is relevant to California vegetable production, as it limits resistance-based management options, and consequently, continuing and future efforts to contain, eradicate, and limit the further distribution or new introductions of this nematode are important.

Table 1 :
Reported host status and reproduction factor (RF) of vegetable crop cultivars for Meloidogyne floridensis.
a NA: not available b -: reproduction factor < 1 or reported as resistant or non-host.+: reproduction factor ≥ 1 or reported as a host plant c Kokalis-Burelle and Nyczepir, 2004 d Marquez and Hajihassani, 2023 e Brito et al., 2008 f Stanley et al., 2009 g Handoo et al., 2004 h Ploeg and Stoddard, 2023 (Continued)

Table 2 :
Average (n=5 for each crop cultivar -Meloidogyne isolate combination) root-galling index (0-10 = no galling -100% of root system with galls; ±standard error) on roots of different plant cultivars 8 weeks after inoculation with 10,000 eggs/plant of a California isolate of M. incognita race 3 (Minc-3) or M. floridensis (Mflor) in two repeated greenhouse pot experiments.
a (S) or (R) indicates a Meloidogyne incognita susceptible or resistant cultivar, respectively.bDifferent letters within rows indicate significant differences at the 95% confidence level, Kruskal-Wallis test.Host status

Table 3 :
Average (n=10 for each crop cultivar -Meloidogyne isolate combination) root-knot nematode reproduction factor RF (number of eggs per root system at harvest/number of eggs inoculated; ±standard error) on different plant cultivars 8 weeks after inoculation with 10,000 eggs/plant of a California isolate of M. incognita race 3 (Minc-3) or M. floridensis (Mflor) in greenhouse pot experiments.Statistical analysis was done on log(x+1)-transformed data.Non-transformed data shown.Different letters within rows indicate significant differences at the 95% confidence level (Fisher's protected LSD test).
a (S) or (R) indicates a Meloidogyne incognita susceptible or resistant cultivar, respectively.b

Table 4 :
Summary of results from this study on host status and reproduction factor (RF) of vegetable crop cultivars for Meloidogyne floridensis.

of crops for Meloidogyne floridensis: Ploeg and Edwards crop
may, over time, shift to a larger proportion of the population belonging to M. incognita.In our study, pepper Carolina Wonder was a suitable host to distinguish M. incognita from M. floridensis.Other hosts that consistently separated M. floridensis from M. incognita include several M. incognita-resistant Prunus rootstocks such as NemaGuard, NemaRed,